Bottom Line:
The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

Affiliation: Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACTPseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

pone.0129629.g005: Phenotypic microarrays of PAOΔmifS and PAOΔmifR mutants.The loss of mifS and mifR results in a growth deficient phenotype in the presence of α-KG as a sole carbon source, as depicted by BioLOG plate PM1, well D6 (A and B). Loss of growth phenotype was confirmed by growing PAO1, PAO∆mifS, PAO∆mifR and PAO∆mifSR mutants in M9 minimal media with α-KG (30 mM) for 18 to 24 h at 37°C (C). The growth defect was rescued by expressing mifR and mifSR genes (D) and the gene encoding the α-KG specific transporter PA5530 (E) in trans.

Mentions:
The inability to utilize α-KG by PAO∆mifS (Fig 5A) and PAO∆mifR (Fig 5B) in the BioLOG assay was reproduced in M9 minimal media supplemented with 30 mM α-KG (Fig 5C). In fact, all three mutant strains, PAO∆mifR, PAO∆mifS and PAO∆mifSR failed to grow in the presence of α-KG (Fig 5C). To rule out potential toxicity, the wild-type P. aeruginosa PAO1 and the mutants were cultured in M9 minimal media with varying concentrations of α-KG, ranging from 1 to 80 mM (Fig 6). The mutants exhibited no growth in the presence α-KG after 24 h at 37°C, whereas the wild-type PAO1 exhibited an increase in growth that was proportional to α-KG concentration (Fig 6B). All subsequent experiments were done with 30 mM α-KG. The growth defect exhibited by PAO∆mifS, PAO∆mifR and PAO∆mifSR could be restored to the wild-type levels by introducing mifR and mifSR genes into the respective mutants (Figs 5D and 7A).

pone.0129629.g005: Phenotypic microarrays of PAOΔmifS and PAOΔmifR mutants.The loss of mifS and mifR results in a growth deficient phenotype in the presence of α-KG as a sole carbon source, as depicted by BioLOG plate PM1, well D6 (A and B). Loss of growth phenotype was confirmed by growing PAO1, PAO∆mifS, PAO∆mifR and PAO∆mifSR mutants in M9 minimal media with α-KG (30 mM) for 18 to 24 h at 37°C (C). The growth defect was rescued by expressing mifR and mifSR genes (D) and the gene encoding the α-KG specific transporter PA5530 (E) in trans.

Mentions:
The inability to utilize α-KG by PAO∆mifS (Fig 5A) and PAO∆mifR (Fig 5B) in the BioLOG assay was reproduced in M9 minimal media supplemented with 30 mM α-KG (Fig 5C). In fact, all three mutant strains, PAO∆mifR, PAO∆mifS and PAO∆mifSR failed to grow in the presence of α-KG (Fig 5C). To rule out potential toxicity, the wild-type P. aeruginosa PAO1 and the mutants were cultured in M9 minimal media with varying concentrations of α-KG, ranging from 1 to 80 mM (Fig 6). The mutants exhibited no growth in the presence α-KG after 24 h at 37°C, whereas the wild-type PAO1 exhibited an increase in growth that was proportional to α-KG concentration (Fig 6B). All subsequent experiments were done with 30 mM α-KG. The growth defect exhibited by PAO∆mifS, PAO∆mifR and PAO∆mifSR could be restored to the wild-type levels by introducing mifR and mifSR genes into the respective mutants (Figs 5D and 7A).

Bottom Line:
The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

Affiliation:
Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACTPseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.